1, Field of the Invention
The present invention is in the field of microfluidics and physiology. More specifically, the invention relates to microfluidic systems that mimic the structure, fluid flow characteristics, and physiological behavior of small physiological vessels such as those found in the microvasculature. The invention has many uses including the optimization of drug delivery and microvascular treatments and in describing disease mechanisms that affect the microvasculature such as inflammation, diabetes and hypertension. The invention is not limited to vessels of the circulatory system but is applicable to all physiological vessels such as lymphatic vessels and glandular ductules.
2, Description of Related Art
Physiological microvascular networks are series of interconnected arterioles, capillaries, and/or venules. The diameters of these vessels range from about 2-10 μm for capillaries and about 10-100 μm for arterioles and venules. In the context of the present invention, the term “microvascular networks” can also be used to describe networks of physiological vessels having diameters of less than about 100 μm such as renal or seminiferous tubules.
A considerable body of work has been developed regarding in-vitro systems for the study of vascular endothelial responses, leukocyte adhesion, and drug carrier delivery in the presence of flow (Blackwell 2001; Burch 2002; Chang 2000; Cozens-Roberts 1990; Crutchfield 2000; Dickerson 2001; Goetz 1994; Kiani 2002; Patel 1999; Patil 2001; Sayed 2001). For example, in-vitro flow chambers have facilitated the identification of biological molecules involved in the adhesion of leukocytes to the endothelium. It is now known that the dynamics of cell adhesion to vascular endothelial cells are controlled by a combination of biochemical and macro-properties such as vessel size and flow rate. These results have led in part to an effort to understand endothelial cell-leukocyte interactions using carefully controlled in-vitro flow cell experiments.
The adhesion of particles such as leukocytes, platelets, liposomes/lipisomes, and microencapsulated drug carriers to microvascular endothelium is also greatly influenced by the geometric features of the vasculature, local hemodynamics, and numerous receptor-ligand interactions between endothelial cells and particles. Local hemodynamic factors associated with microvascular geometry such as wall shear stress, pressure, and residence time influence the rates, amounts, and distributions of particle adhesion as well as endothelial cell morphology and function. This complex interplay between flow, cells, and particles is still poorly understood and it is not possible to predict, for example, adhesion patterns and numbers of adhered particles in the microvasculature based on current in-vitro flow cell technologies. The present invention advances in-vitro flow cell technology so that adhesion patterns and numbers of adhered particles in the microvasculature, for example, can be predicted.
In-vitro flow chambers typically comprise a single flow channel formed by two plates or slides separated by a gap. The cross section of the flow channel is typically a flat rectangle with constant dimensions. The flow chambers are usually transparent and are perfused at low Reynolds numbers to match wall shear stresses observed in blood vessels in-vivo. The lower plates of these flow chambers are coated with either protein or adherent endothelial cells to simulate the surface properties of physiological vessels (Luscinskas 1994). The chambers are typically mounted on a microscope stage and events are recorded with a high-speed camera and stored for subsequent analysis.
Recently, efforts to construct microfluidic flow chambers have been reported. These efforts typically feature linear channels with conventional rectangular cross sections and constant cross sectional areas. For example, Lu et al. (2004) reports the fabrication of a device on PDMS for varying shear profiles. Unlike the present invention, this and other existing device do not account for the geometric variations and the interconnectedness found in microvascular networks and lack the ability to effectively reproduce or simulate the flow patterns and particle adhesion patterns observed in physiological vessels and microvasculature.
The smallest cross section of commercially available in-vitro flow chambers is typically about 2500 μm×125 μm, which is significantly larger than arterioles, capillaries, and venules. They do not provide realistic sizes and geometries corresponding to complex combinations of thoroughfare channels, bifurcations, junctions, convolutions, and/or variable diameters found in in-vivo microcirculation. These complex features determine local fluid dynamic profiles and thereby local values of shear stress, pressure, residence time, and velocity. Complex geometrical features also strongly influence the transport and adhesion of cells and other particles to sites in the microvasculature. In diseased networks such as those affected by tumor growth, stenoses, arteriosclerosis, diabetes, and radiation therapy, both flow profiles and microvasculature features are different from healthy networks. Examples include medullary arteriolar tortuosity seen in hypertension and intraparenchymal arteriolar-to-arteriolar anastomoses in pathological conditions in the cerebrum. Existing in-vitro flow chambers, however, having idealized rectangular or circular duct geometries cannot be modified to reflect changes that occur with disease.
Some attempts have been made to improve on the scale of in-vitro microvascular channels. Cokelet et al. (1993) fabricated an in-vitro microvascular channel to study blood flow by photolithography on microscope slide glass. Frame et al. (1995) generated idealized (straight) semi-circular microvascular channels (20-50 μm) on glass to mimic the arteriolar microcirculation. They were also successful in growing endothelial cells on these channels. However, both studied idealized (straight) microchannels that did not account for either the interconnectedness or variations found in in-vivo microvascular networks.
The complexity of the flow in microvascular networks necessitates the use of sophisticated models to analyze and understand flow behavior. Mathematical modeling has been used extensively to study particle/cell adhesion in a flow environment. Starting with the pioneering work of Hammer & Lauffenburger (1987), Cozens-Roberts et al. (1990) derived an analytical expression for the shear stress and adhesive force relationships in flow chambers. Hammer and coworkers (1992) developed mathematical models for cell attachment and rolling. The dependence of rolling adhesion of cells on vessel diameters and other parameters has been addressed in several notable works including, Goldsmith & Turitto (1986), Schmid-Schoenbein et al. (1975), House & Lipowsky (1988) and Chapman & Cokelet (1998). The biochemical processes of receptor-ligand attachment have been extensively studied as well (Goetz 1994; Hammer 1992). Chapman & Cokelet (1993, 1998) used computational fluid dynamics modeling (CFD) to analyze the hemodynamic impact of leukocytes adherent to the wall of post-capillary venules. Chang et al. (2000) disclosed an “Adhesive Dynamics” technique for studying and developing a state diagram for cell adhesion in flow, under simple conditions.
All of the numerical studies mentioned above are based upon simple, idealized flow chambers with regular geometries and have not examined flow profiles and shear stresses in realistic in-vivo microvasculature scenarios with relevance to cell adhesion. In contrast, the present invention includes a CFD-based model framework that analyzes fluid flow and particle motion/adhesion in the context of realistic in-vivo networks that correspond to the fabricated vascular networks of the invention. The CFD models can be used to analyze and interpret results from experiments and to convert them into information used to assist with the design of experiments and treatment protocols.
In summary, there remains a need in the art for an in-vitro flow chamber that accurately simulates the anatomical and hemodynamic properties of physiological microvascular networks. There is also a need for methods of using such a flow chamber that describe and predict the behavior of particles and cells in microvascular networks. Such a flow chamber and methods can be used, for example, to screen materials and methods for optimal drug delivery to both healthy and diseased microvasculature.